The epigenome as a putative target for skin repair: the HDAC inhibitor Trichostatin A modulates myeloid progenitor plasticity and behavior and improves wound healing.

BACKGROUND: The molecular pathways that drive bone marrow myeloid progenitors (BMMP) development are very well understood and include a tight controlled multi-stage gene hierarch. Monocytes are versatile cells that display remarkable plasticity and may give rise to specific subsets of macrophages to proper promote tissue homesostasis upon an injury. However, the epigenetic mechanisms that underlie monocyte differentiation into the pro-inflammatory Ly6Chigh or the repairing Ly6Clow subsets are yet to be elucidated. We have previously shown that Epigenetic mechanisms Histone Deacetylase (HDAC) dependent are crucial for monocyte behavior and plasticity and in this work, we propose that this same mechanism underlies BMMP plasticity upon an inflammatory challenge in vivo. METHODS: BMMP were culture in the presence of GM-CSF alone or in combination with HDAC inhibitor (iHDAC) and phenotyped by flow cytometry, immune staining or western blot. iHDAC was topically added to skin wounds for 7 consecutive days and wound healing was monitored by flow cytometry and histopathological analysis. RESULTS: When BMMP were cultured in the presence of iHDAC, we showed that the CD11blow/Ly6Clow subset was the specific target of iHDAC that underwent chromatin hyperacetylation in vitro. Upon 13 days in the presence of iHDAC, BMMP gave rise to very elongated macrophages, that in turn, displayed a remarkable plasticity in a HDAC activity dependent fashion. HDAC-dependent cell shape was tight related to macrophage behavior and phenotype through the control of iNOS protein levels, showing that chromatin remodeling is a key component of macrophage plasticity and function. We then hypothesized that iHDAC would modulate the inflammatory response and favor tissue repair in vivo. To test this hypothesis, we topically added iHDAC to skin wounds during 7 consecutive days and followed tissue repair dynamics. In fact, iHDAC treated skin wounds presented an increase in wound closure at day 5 that was correlated to an enrichment in the CD11blow/Ly6Clow subset and in very elongated F4/80 positives macrophages in vivo, fully recapitulating the behavior previously observed in vitro. CONCLUSION: Our work provides the biological basis that connects chromatin remodeling to phenotypic plasticity, which in turn, may become a tractable therapeutic strategy in further translational studies.

Immune and metabolic shifts during neonatal development reprogram liver identity and function.

BACKGROUND & AIMS: The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. METHODS: We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. RESULTS: Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. CONCLUSION: In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. LAY SUMMARY: Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.

Persistence of Brucella abortus in the Bone Marrow of Infected Mice.

Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.

Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice.

This work evaluated the capacity of two probiotic strains, Lactobacillus casei CRL431 and Lactobacillus rhamnosus CRL1506, to protect against myelosuppression and immunosuppression in cyclophosphamide (Cy)-treated mice. Changes in mature granulocytes and progenitor cells in bone marrow (BM) and blood were studied. In addition, the ability of probiotics to accelerate the recovery of the immune response against the opportunistic pathogen Candida albicans was evaluated. We demonstrated for the first time that the preventive treatment with immunomodulatory lactobacilli such as L. casei CRL431 or L. rhamnosus CRL1506 was able to increase immature myeloid progenitors in the BM, allowing an early recovery of myeloid cells after Cy administration. Probiotic lactobacilli were also capable to induce an early recovery of neutrophils in blood, improve phagocytic cells recruitment to infectious sites and increase the resistance against the opportunistic pathogen C. albicans. Although deeper studies regarding the cellular and molecular mechanisms of probiotic actions are needed, these findings support the idea that strains like CRL431 and CRL1506 may accelerate the recovery of Cy-caused immunosuppression by immunopotentiating myeloid cells. Then, probiotic lactobacilli have the potential to be used as alternatives for lessening chemotherapy-induced immunosuppression in cancer patients.

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion.

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increased CD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

ß-1,3-Glucan given orally modulates immunomyelopoietic activity and enhances the resistance of tumour-bearing mice.

ß-Glucans have been reported to be potent adjuvants in stimulating innate and adaptive immune responses. The aim of the present study was to determine the immunohematopoietic effects of Imunoglucan (HEBRON) following its oral administration to normal and Ehrlich ascites tumour (EAT)-bearing mice. Mice were treated with 250, 500 and 1000 mg/kg per day, p.o., Imunoglucan (ß-1,3-glucan extracted from Saccharomyces cerevisae) for 18 consecutive days. Treatment started 10 days prior to and ended 8 days after tumour inoculation. At 500 and 1000 mg/kg per day, Imunoglucan enhanced the life span of EAT-bearing mice and prevented myelosuppression and splenomegaly caused by the tumour by increasing the number of granulocyte-macrophage progenitors in the bone marrow and increasing colony-stimulating activity in the serum. At 500 mg/kg, Imunoglucan restored the reduced ability of stromal cells to display myeloid progenitors in long-term bone marrow cultures of EAT-bearing mice and upregulated the production of interleukin (IL)-6 and IL-1α by these cells, consistent with a higher number of non-adherent cells. Moreover, 500 mg/kg Imunoglucan restored natural killer cell activity in tumour-bearing mice, consistent with the increased production of interferon (IFN)-γ observed. The results of the present study suggest that Imunoglucan given orally indirectly modulates immune activity and probably disengages tumour-induced suppression by producing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (colony-stimulating factors, IL-1α, IL-6 and IFN-γ).

Uncaria tomentosa stimulates the proliferation of myeloid progenitor cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable. It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology. MATERIALS AND METHODS: The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 µg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB). RESULTS: Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 µg extract/mL. CONCLUSIONS: At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a 'powerful plant'.

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines.

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte-macrophage progenitors (GMPs), whereas differentiation into megakaryocyte-erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1(+)Mac-1(+) myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca(2+) chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte-monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.

[Cancer immunotherapy. Importance of overcoming immune suppression]. / Inmunoterapia del cáncer. Importancia de controlar la inmunosupresión.

Increasing evidence indicates that the immune system is involved in the control of tumor progression. Effective antitumor immune response depends on the interaction between several components of the immune system, including antigen-presenting cells and different T cell subsets. However, tumor cells develop a number of mechanisms to escape recognition and elimination by the immune system. In this review we discuss these mechanisms and address possible therapeutic approaches to overcome the immune suppression generated by tumors.

Inmunoterapia del cáncer: Importancia de controlar la inmunosupresión / Cancer immunotherapy: Importance of overcoming immune suppression

Es cada vez mayor la evidencia experimental y clínica de que el sistema inmunitario interviene activamente en la patogénesis y el control de la progresión tumoral. Una respuesta antitumoral efectiva depende de la correcta interacción de varios componentes del sistema inmunitario, como las células presentadoras de antígeno y diferentes sub-poblaciones de linfocitos T. Sin embargo, los tumores malignos desarrollan numerosos mecanismos para evadir el reconocimiento y su eliminación por parte del sistema inmunitario. En esta revisión discutiremos algunos de esos mecanismos y posibles estrategias terapéuticas para contrarrestarlos.

Increasing evidence indicates that the immune system is involved in the control of tumor progression. Effective antitumor immune response depends on the interaction between several components of the immune system, including antigen-presenting cells and different T cell subsets. However, tumor cells develop a number of mechanisms to escape recognition and elimination by the immune system. In this review we discuss these mechanisms and address possible therapeutic approaches to overcome the immune suppression generated by tumors.

A human dendritic cell subset receptive to the Venezuelan equine encephalitis virus-derived replicon particle constitutively expresses IL-32.

Dendritic cells (DCs) are a diverse population with the capacity to respond to a variety of pathogens. Because of their critical role in pathogenesis and Ag-specific adaptive immune responses, DCs are the focus of extensive study and incorporation into a variety of immunotherapeutic strategies. The diversity of DC subsets imposes a substantial challenge to the successful development of DC-based therapies, requiring identification of the involved subset(s) and the potential roles each contributes to the immunologic responses. The recently developed and promising Venezuelan equine encephalitis replicon particle (VRP) vector system has conserved tropism for a subset of myeloid DCs. This immunotherapeutic vector permits in situ targeting of DCs; however, it targets a restricted subset of DCs, which are heretofore uncharacterized. Using a novel technique, we isolated VRP-receptive and -nonreceptive populations from human monocyte-derived DCs. Comparative gene expression analysis revealed significant differential gene expression, supporting the existence of two distinct DC populations. Further analysis identified constitutive expression of the proinflammatory cytokine IL-32 as a distinguishing characteristic of VRP-receptive DCs. IL-32 transcript was exclusively expressed (>50 fold) in the VRP-receptive DC population relative to the background level of expression in the nonreceptive population. The presence of IL-32 transcript was accompanied by protein expression. These data are the first to identify a subset of immature monocyte-derived DCs constitutively expressing IL-32 and they provide insights into both DC biology and potential mechanisms employed by this potent vector system.

Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation.

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. G(M3) was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor alpha chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous G(M3). In these cells, G(M1) was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor alpha chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.

Uncaria tomentosa extract increases the number of myeloid progenitor cells in the bone marrow of mice infected with Listeria monocytogenes.

In this study, we demonstrated that Uncaria tomentosa extract (UTE) protects mice from a lethal dose of Listeria monocytogenes when administered prophylactically at 50, 100, 150 and 200 mg/kg for 7 days, with survival rates up to 35%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with L. monocytogenes, due to increased numbers of granulocyte-macrophage progenitors (CFU-GM) in the bone marrow. Non-infected mice treated with 100 mg/kg UTE also presented higher numbers of CFU-GM in the bone marrow than the controls. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected mice pre-treated with UTE. Moreover, stimulation of myelopoiesis and CSA occurred in a dose-dependent manner, a plateaux being reached with the dose of 100 mg/kg. Further studies to investigate the levels of factors such as IL-1 and IL-6 were undertaken. We observed increases in the levels of IL-1 and IL-6 in mice infected with L. monocytogenes and treated with 100 mg/kg of UTE. White blood cells (WBC) and differential counting were also performed, and our results demonstrated no significant changes in these data, when infected mice were pre-treated with 100 mg/kg of UTE. All together, our results suggest that UTE indirectly modulates immune activity and probably disengages Listeria-induced supression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1 and IL-6).

Effects produced by Royal Jelly on haematopoiesis: relation with host resistance against Ehrlich ascites tumour challenge.

Royal jelly (RJ) was shown to exhibit immunomodulatory properties, although its biological activity is still unclear. In order to elucidate the mechanism whereby RJ activates the immunological system, we examined the role of this substance on the haematopoietic response of Ehrlich ascites tumour (EAT)-bearing mice. Our results demonstrated that RJ prevented the myelosupression induced by the temporal evolution of the tumour and abrogated the splenic haematopoiesis observed in EAT-bearing mice. The stimulating effect of RJ was also observed in vitro on the multipotent bone marrow stem cells, evaluated by the long-term bone marrow cultures (LTBMCs). The study of survival clearly showed the antitumour activity of RJ. Treatment was given prophylactically for 20 days and therapeutically for 3, 8 and 13 days. Except for the treatment with the lower dose of 500 mg/kg, given for 23 days, all the other dose schedules were able to prolong survival. A more effective antitumoural response was observed with the more prolonged treatment regimen. In this regard, the administration of RJ for 33 days produced the highest protection reaching an extension of survival at about 38%, 71% and 85% for the doses of 500, 1000 and 1500 mg/kg, respectively, whereas with the 23 and 28 days treatment schedules, survival increased at a rate of 19% and 23%, respectively, and comparable results were found among the effective doses of RJ. Increased survival rate might be related to the decreased Prostaglandin E2 (PGE2) levels observed in EAT-bearing mice after RJ treatment. These results point to RJ as a promising modifier of biological response leading to myeloprotection and antitumour activity.

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.

The capacity of connective tissue stromas to sustain myelopoiesis depends both upon the growth factors and the local intercellular environment.

In adults, haemopoiesis is located in the bone marrow, where it is tightly regulated by cytokines and by a physical association of haemopoietic progenitors with the stroma. However, in pathological situations, haemopoiesis can be partly or fully dislodged to peripheral tissues. It is not clear which are the requirements for a given peripheral stroma to sustain haemopoiesis. Using the growth factor-dependent cell line FDC-P1, we have compared the myelopoietic capacities of a murine bone marrow-derived cell line S17, a liver inflammatory granuloma-derived stroma (GR) that sustains haemopoiesis, and normal skin fibroblasts (SF) that sustain neither survival nor proliferation of myeloid cells. All three stromas expressed mRNA for major haemopoietins with the exception of IL-3. Despite the incapacity of SF to sustain FDC-P1 cells, the biologically active GM-CSF could be recovered from all the studied stromas by treatment with high-salt buffers that release non-covalently bound molecules from stroma cells. Glycosaminoglycans purified from stromas had distinct effect on the GM-CSF-mediated proliferation of FDC-P1 cells: those purified from S17 and GR cells were stimulatory, whereas those obtained from SF cells were slightly stimulatory at low concentration, but inhibitory at the higher ones. We conclude that the quality of the stroma pericellular glycoconjugates is determinant for the ability of a given stroma to sustain myelopoiesis, even when biologically active haemopoietins are locally produced.

Efeito da prostaglandina E2 sobre a resposta de precursores de eosinófilos e progenitores mielóides da medula óssea murina à IL-5 e GM-CSF / The effect of prostaglandin E2 on the response of eosinophil precursors and bone marrow myeloid progenitors murine IL-5 and GM-CSF

Avaliamos o efeito da prostaglandida E2 (PGE2) sobre precursores e progenitores murinos, em resposta à interleucina 5(IL-5) ou fator estimulador de colônias de granulócitos e macrófagos (GM-CSF), respectivamente. Precursores foram estudados em cultura líquida de medula óssea e progenitores foram analisados em teste de formação de colônias. As respostas de camundongos normais e de camundongos sensibilizados com ovalbumina, e provocados por via intranasal, foram comparadas...Nas outras linhagens isogênicas de camundongos (A/J, CBA/J, C57/BL10) os progenitores mielóides responderam ao tratamento pela PGE2 (10 M). Progenitores mielóides de camundongos F1 (C57/BL10 x BALB/c) também se mostraram sensíveis à PGE2 (10 M), indicando que resistência de camundongos BALB/c deve ser um defeito recessivo. Estes resultados demonstram, pela primeira vez, um efeito inibitório da PGE2 sobre a eosinopoiese, e apontam para progenitores e precursores como alvos distintos para a regulação pela PGE2.